There has been an explosive increase in our understanding of RNA structure and ground breaking advances in defining the roles of RNAs in gene expression and regulation. Yet, our understanding of the chemical and biophysical properties of RNA that determine its biological function has advanced less quickly. This deficiency is due to the complexity of the problem, but also due to the lack of sufficient experimental tools for revealing mechanistic detail. The ribonucleoprotein enzyme ribonuclease P (RNase P), which catalyzes the essential 5' end maturation of tRNA precursors (ptRNAs), has emerged as an elegantly simple and broadly useful system to understand RNA structure and function including catalysis. The long term goal of our project is to understand, at a chemical level of detail, how the RNase P ribonucleoprotein achieves its enormous rate enhancement and its multiple substrate specificity. Essential to both processes is site-specific binding of Mg2+ ions. Integrated into our experimental analyses of RNase P are innovative research tools designed to overcome key experimental limitations in three areas: defining RNA-metal ion interactions (Raman spectroscopy); identifying catalytic interactions (kinetic isotope effects); and understanding multiple substrate recognition (high-throughput sequencing). RNase P, like many enzymes, processes multiple different substrates in the cell. This property raises the general problem of how the enzyme distinguishes between cognate and non-cognate substrates and how it accommodates the variation in structure between different substrates. We are comparing the kinetics of different ptRNA processing reactions, and applying a novel high-throughput method to identify the ptRNA sequences P that control optimal catalytic efficiency. Despite intense investigation, the catalytic modes employed by ribozymes, including RNase P, are not well understood or characterized experimentally. We are pursuing detailed mechanistic analyses to test proposed active site interactions by observing how site-specific functional group modifications in P RNA and ptRNA influence the charge distribution in the transition state. The interaction of solution Mg2+ ions is essential for the function of all RNAs, and establishing the relationships between the binding of individual ions or classes of ions is an area of intense interest. However, like most RNAs the linkages between individual ion interactions in P RNA, and the critical enzyme functions of binding and catalysis are not well understood. In the last project period, we developed a means to detect and quantify metal ion interactions with RNA phosphates using Raman spectroscopy. To follow up on these advances we are using Raman spectroscopy and direct ion association measurements to detect the uptake of ions upon formation of the ES complex and to test the roles of P4 residues in ion binding. Additionally we are developing methods to detect ion binding at individual phosphates using isotope-edited Raman. PUBLIC HEALTH RELEVANCE: Cell function depends on complexes of RNA and protein to process RNAs and synthesize proteins, and these RNA-protein complexes are the target of numerous diagnostics, drugs and clinical therapies. The research will for the first time test directly how an essential RNA-protein complex called RNase P recognizes its substrate and accomplishes catalysis during an essential step in RNA biosynthesis.